Retinal pigment epithelial cells differentiated from embryonic stem cells with nicotinamide and activin A
The present invention concerns RPE cells obtainable by directed differentiation from stem cell, particularly, human stem cells. It has been specifically found that culturing stem cells in the presence of one or more member of the TGFβ superfamily, such as Activin A) induced directed differentiation into mature and functional RPE cells. This was evidenced by the expression of markers specific to mature RPE cells, including MiTF-A, RPE65 or Bestrophin). In accordance with one particular embodiment, the cells are a priori cultured with nicotinamide (NA) which was found to augment the cells' response to the inductive effect of the one or more member of the TGFβ superfamily. The invention also provides methods of performing the directed differentiation, as well as methods for use of the resulting RPE cells.
1. A method for promoting directed differentiation of human pluripotent stem cells into retinal pigment epithelium (RPE) fate, the method comprising:
(a) culturing human pluripotent stem cells in a medium supplemented with nicotinamide (NA) to generate differentiating cells; and subsequently
(b) culturing said differentiating cells in a medium supplemented with activin A under conditions which induce said differentiating cells to differentiate into RPE cells.
2. The method of claim 1 , wherein said culturing in said medium supplemented with NA is effected for at least two days prior to culturing in said medium supplemented with said activin A.
3. The method of claim 1 , wherein said cells are cultured in a suspension.
4. The method of claim 1 , wherein said human pluripotent stem cells are human embryonic stem cells.
5. The method of claim 1 , wherein culturing said differentiating cells in said medium supplemented with nicotinamide is effected for about two weeks.
6. The method of claim 1 , wherein culturing said differentiating cells in said medium supplemented with activin A in step (a) is effected for about two weeks.
7. The method of claim 1 , wherein a concentration of said nicotinamide is about 10 mM.
8. The method of claim 1 , wherein a concentration of said activin A is between 20-180 ng/ml.
9. The method of claim 1 , wherein said medium supplemented with activin A is further supplemented with nicotinamide.